Despite long-term suppression of HIV-1 viral load in plasma to undetectable levels, HIV-1 has not been eradicated from patients treated with potent antiretroviral regimens. The available evidence suggests this inability to eradicate HIV-1 infection is due to ongoing low-level viral replication and persistent reservoirs of infection despite potent therapy. We posit that extra-chromosomal circular HIV DNA (DNA circles), which is detectable in peripheral blood monocytic cells (PBMC) despite long-term suppression of HIV viral load in plasma to undetectable levels, is a i marker for continued viral replication in these patients and may demonstrate the source(s) of persistent viral replication and potential persistent reservoirs of infection. In this application, we propose 3 studies to test these hypotheses and to try to answer these basic questions about HIV-1 pathogenesis: 1) Evolution of HIV sequences occurs during HIV-1 replication and so is a marker for ongoing viral replication. We will evaluate for sequence evolution over time between HIV DNA circles (representing new cellular infection) and integrated proviral DNA (archival sequence) in PBMC from patients who have had long-term suppression of viral replication with potent antiretroviral regimens; 2) Despite long-term suppression of plasma HIV-1 viral load to undetectable levels with potent antiretroviral medication, upon discontinuation of the medication, plasma viremia quickly returns. The exact source of this "rebounding" virus is not known, however. We will therefore determine the genetic relatedness between the low-level replicating virus found in PBMC during long-term antiretroviral therapy, which we posit is the main source of the "rebounding" virus (represented by DNA circles) versus the virus that rebounds after discontinuation of antiretroviral therapy, and further, in comparison with proviral DNA sequences found in seminal mononuclear cells, a putative persistent reservoir of infection; and 3) The sub-population(s) of lymphocytes that are infected during ongoing low-level HIV-1 replication in patients receiving potent antiretroviral therapy have not been identified. Identification of these newly infected lymphocytes would supply clues as to the origin of the persistent reservoirs of infection in these patients. We will therefore combine cell sorting techniques with our assay for DNA circles to identify specific sub-population(s) within PBMC and seminal mononuclear cells that harbor DNA circles. Identification of the reservoirs of persistent HIV-1 infection is crucial for understanding the mechanisms of persistence of HIV-1 infection in patients treated with potent antiretroviral medications and may lead to the development of new therapeutic agents and approaches for the eradication of HIV-1 from infected patients. These studies will also provide me with the opportunity to develop the required skills to reach my long-term goal: a career as an independent investigator and academician performing patient-oriented translational research.